There is significant interest in the development of targeted therapeutics such as antibody drug conjugates that have the potential to improve the therapeutic window of cytotoxic drugs by delivering them specifically and intracellularly to cancer cells (Austin et al. (2004) Mol. Biol. Cell. 15: 5268-5282; Burris et al. (2011) Clin. Breast Cancer. 11: 275-282; Sievers and Senter (2013) Annu. Rev. Med. 64: 15-29; Behrens and Liu (2013) MAbs, 6(1): 46-53; Sutherland et al. (2006) J. Biol. Chem. 281: 10540-10547). The pathway by which the targeted agent enters tumor cells can influence both the uptake efficiency and the intracellular fate of the internalized agent, both of which contribute to the cytotoxic potency (Sutherland et al. (2006) J. Biol. Chem. 281: 10540-10547; Erickson et al. (2006) Cancer Res. 66: 4426-4433).
Endocytosis pathways can be subdivided into four categories: 1) clathrin-mediated endocytosis, 2) caveolae, 3) macropinocytosis, and 4) phagocytosis. Clathrin-mediated endocytosis is mediated by small (approx. 100 nm in diameter) vesicles that have a morphologically characteristic coat made up of a complex of proteins that are mainly associated with the cytosolic protein clathrin. Clathrin-coated vesicles (CCVs) are found in virtually all cells and form domains of the plasma membrane termed clathrin-coated pits. Coated pits can concentrate large extracellular molecules that have different receptors responsible for the receptor-mediated endocytosis of ligands, e.g. low density lipoprotein, transferrin, growth factors, antibodies and many others.
Caveolae are the most common reported non-clathrin-coated plasma membrane buds, which exist on the surface of many, but not all cell types. They consist of the cholesterol-binding protein caveolin (Vip21) with a bilayer enriched in cholesterol and glycolipids. Caveolae are small (approximately 50 nm in diameter) flask-shape pits in the membrane that resemble the shape of a cave (hence the name caveolae). They can constitute up to a third of the plasma membrane area of the cells of some tissues, being especially abundant in smooth muscle, type I pneumocytes, fibroblasts, adipocytes, and endothelial cells (Burris et al. (2011) Clin. Breast Cancer. 11: 275-282). Uptake of extracellular molecules is also believed to be specifically mediated via receptors in caveolae.
Macropinocytosis, which usually occurs from highly ruffled regions of the plasma membrane, is the invagination of the cell membrane to form a pocket, which then pinches off into the cell to form a vesicle (˜0.5-5 μm in diameter) filled with a large volume of extracellular fluid and molecules within it (equivalent to ˜100 CCVs). The filling of the pocket occurs in a non-specific manner. The vesicle then travels into the cytosol and fuses with other vesicles such as endosomes and lysosomes.
Phagocytosis is the process by which cells bind and internalize particulate matter larger than around 0.75 μm in diameter, such as small-sized dust particles, cell debris, micro-organisms and even apoptotic cells, which only occurs in specialized cells. These processes involve the uptake of larger membrane areas than clathrin-mediated endocytosis and caveolae pathway.